Date of Award

8-2026

Document Type

Thesis

Degree Name

Master of Science in Biology

Department

Biology

First Reader/Committee Chair

Bournias-Vardiabasis, Nicole

Abstract

Neural crest cells (NCCs) are a transient, multipotent cell population that contributes to craniofacial structures, peripheral neurons, melanocytes, and smooth muscle. Defects in NCC development underlie a broad range of congenital disorders known as neurocristopathies. Human pluripotent stem cell (hPSC)-derived NCCs provide an evaluable model for studying early human development; however, existing differentiation protocols produce variable outcomes, and no standardized method consistently generates high-fidelity NCC populations. To address this challenge, we compared five published hPSC-to-NCC differentiation protocols using the H9 human embryonic stem cell line. We hypothesized that specific protocols more faithfully recapitulate neural crest development through coordinated regulation of Wnt, BMP, and TGF-β signaling. In this study, NCC populations were evaluated for NCC phenotypic markers using immunocytochemistry and flow cytometry. Furthermore, molecular fidelity was assessed using targeted-qPCR. These two scoring methods were combined for an overall Integrated Fidelity Scoring. The Avery protocol had the highest Integrated Fidelity Score, and provided the highest-fidelity recapitulation of neural crest induction (Figure 18). However, the Duarte protocol had the strongest phenotypic neural crest resemblance, and the Kreitzer protocol had the strongest molecular fidelity alongside Avery (Figure 18). These differences generate developmental states along the neural crest induction pipeline. By standardizing neural crest induction protocols, in vitro NCCs can more faithfully recapitulate in vivo NCCs. Likewise, the quantification and scoring of neural crest differentiation protocols through this novel Integrated Fidelity Scoring system can be utilized as a standard in the fields of developmental biology, regenerative medicine, and developmental toxicology.

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